EL MERCADO EN COSTA RICA

Objective

The brain is essential in regulating intake of food and beverages, by balancing energy- homeostasis, regulated by the hypothalamus, with reward perception, regulated by the ventral tegmental area (VTA). The main aim of the current study was to investigate the effects of glucose, fructose, sucrose and sucralose (a non-caloric artificial sweetener) ingestion on the magnitude and trajectory of the hypothalamic and the VTA BOLD responses.

Design

In five visits, 16 healthy men between 18-25 years with a BMI between 20-23 kg/m2

drank five interventions in a randomized order while an functional MRI scan was made. The interventions consisted of: 50g of glucose, fructose, sucrose, or 0.33 grams of sucralose dissolved in 300ml tap water, 300ml of plain tap water was used as the control condition. Blood oxygen level dependent (BOLD) signals were determined in the hypothalamus and the VTA within a manually drawn region of interest. Differences in changes in BOLD signal between stimuli were analyzed using mixed models.

Results

Compared to the control condition, we found a decrease in BOLD signal in the hypothalamus after ingestion of glucose (p=0.0003) and a lesser and but delayed BOLD response after sucrose (p=0.006) and fructose (p=0.003) ingestion. Sucralose led to a smaller and transient response from the hypothalamus (p=0.026). In the VTA, sucralose led to a very similar response to the water control condition leading to an increase in VTA BOLD activity that continues over the measured time period. The natural sugars appeared to only lead to a transient increase in VTA activity.

Conclusions

Glucose induces a deactivation in the hypothalamus immediately after ingestion that continued over following 12 minutes, which is correlated with satiety signaling by the brain. Fructose and sucrose are both associated with a delayed and lesser response from the hypothalamus, likely because the sugars first have to metabolized by the body. Sucralose leads to the smallest and transient decrease in BOLD in the hypothalamus and leads to a similar response as plain water in the VTA, which indicates that sucralose might not have a similar, possibly satiating, effect on the brain as the natural sugars.

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INTRODUCTION

Our brain is essential in regulating intake of food and beverages, by balancing energy- homeostasis with reward perception [1]. The hypothalamus is an important structure that regulates energy homeostasis by integrating information from glucose and insulin trajectories, with varying levels of hormones and peptides from the gut and stomach [2-5]. The mesolimbic pathway, together with the homeostatic regulation, is responsible for the hedonic response to food. The ventral tegmental area (VTA) and other areas of the limbic system (amygdala, nucleus accumbens) are important parts of the mesolimbic pathway involved in this hedonic response [6]. The VTA is the origin of dopaminergic neurons and dopamine signaling in the mesolimbic system, which is a key substrate for reward prediction and response [7]. The VTA is anatomically and functionally connected to the hypothalamus and integrates homeostatic signals with reward responses [8-10]. Both homeostatic and mesolimbic pathways respond to glucose, which is the natural and preferred source of energy for the body and brain [11, 12]. Glucose concentration in the blood is kept under tight homeostatic control, partly mediated by glucose- sensing neurons in the brain [12]. Glucose intake leads to changes in hypothalamic Blood Oxygen Level Dependent (BOLD) levels, which have been interpreted as a sign of satiety [4, 13]. However, little VTA data are available [14], and further investigation into the VTA may be essential in the understanding of the integration of homeostatic and hedonic responses regulation feeding behavior [6]. In many foods and beverages mono- and di-saccharides or artificial sweeteners are used as sweetener [15]. Increased consumption of these sugars and sweeteners in the modern diet has been speculated to play a role in pathophysiology of obesity, decreased vascular health, metabolic syndrome and type-2 diabetes [15-18]. High fructose consumption has also been hypothesized to have detrimental health effects and is associated with fatty liver disease and has been shown to have greater adverse effects on metabolism and vascular health than glucose in several animal studies [19, 20]. Fructose is used as a sweeter alternative to glucose, allowing for the use of smaller amounts, but the metabolism of both sugars is very different. In contrast to glucose, fructose cannot be used directly as a source of energy [21]. Fructose, which is a naturally occurring saccharide in fruits and vegetables, has to be metabolized by the liver first, before it can be made available as a source of energy [22]. Fructose can be consumed in its free form as a monosaccharide in fruits and high fructose corn syrup but also in the form of sucrose, which is a glucose-fructose disaccharide. In addition to the use of added natural sugars, non-caloric artificial sweeteners are increasingly being used to sweeten foods and beverages. The use of non-caloric sweeteners could be expected to decrease total caloric intake and might therefore be useful to control obesity [23].

However, some epidemiological studies suggest that non-caloric sweeteners might have the opposite effect and might actually lead to increased energy intake [5, 24, 25]. Although these results are not conclusive and the effects of non-caloric sweeteners remain a subject of debate, these result do indicate that these sweeteners could have unexpected effects in the brain. Currently, the homeostatic and mesolimbic effects in the brain of these sweeteners and other common dietary sugars are unknown. The differences in how various sugars are metabolized and made available as energy, and because of the decoupling of sweetness and energy in non-caloric sweeteners, each could lead to different metabolic and physiological responses in the brain [26-29]. To gain a better understanding of the different homeostatic and hedonic responses after ingestion of caloric and non-caloric sweeteners, we investigated the effects of glucose, fructose, sucrose and sucralose (an artificial non-caloric sweetener) on the trajectory and magnitude of BOLD response of the hypothalamus and VTA in normal weight young men.

METHODS

Subjects

Sixteen healthy men aged 18-25 years were recruited by advertisements around Leiden University. Inclusion criteria were: a body mass index (BMI) between 20-23 kg/m2 _and

height between 170-190 cm. Exclusion criteria were: diabetes or a history of disturbances of glucose metabolism; genetic or psychiatric disease affecting the brain; renal or hepatic disease; any chronic disease; weight changes of more than 3 kg within the last 3 months or attempts to lose weight; smoking (within the last 6 months); alcohol consumption of more than 21 units per week; use of recreational drugs (within the last 12 months); recent blood donation; recent participation in other biomedical research projects and contra-indications for MRI scanning. Informed consent was obtained from all participants and the study was approved by the local institutional review board and registered at Clinicaltrails.gov under number NCT03247114. Subject characteristics are shown in table 1.

Table 1. Subject characteristics

Mean ± SD (N=16)

Age (years) 22.4 ± 1.3

Height (m) 1.82 ± 0.06

Weight (kg) 73.0 ± 7.1

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Study design

In this randomized double blind crossover study the participants received five different conditions, one per occasion with a one week interval. Participants were asked to fast for a minimum of 10hs overnight (except for drinking water), while scans were obtained the next morning. Participants received 1 of the 5 conditions on each occasion in a randomized order. The interventions consisted of: glucose, 50g in 300ml tap water; fructose, 50g in 300ml tap water; sucrose, 50g in 300ml tap water; sucralose, 0.33 gr (matched for sweetness with the glucose solution) in 300ml tap water and 300 ml plain tap water as a control condition, no additional flavoring was added to the stimuli. The 50 grams of sugar in our study stimuli is comparable to sugar amounts found in several high energy beverages and was chosen to provide a strong blood glucose and insulin response. The glucose, sucrose and fructose solutions all contained 200 kilocalories, the sucralose sweetened solution was zero kilocalories. All stimuli were ingested at room temperature. Drinking was performed through an oral tube, lying supine in the scanner during the fMRI scan while the participants were monitored by the investigators. Five minutes after the start of the scan subjects were instructed to drink the total amount in a steady and continuous way. Scanning was continued during and after the start of the ingestion for 16 minutes, making for a total scan time of 21 minutes with five minutes pre-ingestion, four minutes ingestion and twelve minutes post-ingestion.

Blood samples and Visual Analogue scales

Before and after the scanning procedure, 5 ml blood samples were taken by venipuncture in an antecubital vein. Insulin and glucose levels before and 30 minutes after ingestion of the study stimuli were determined by the laboratory for Clinical Chemistry at Leiden University Medical Centre. Plasma glucose was measured using a fully automated Hitachi 704/911 system. Plasma insulin was measured by a radioimmunoassay (Medgenix, Fleurus, Belgium). Participants were asked to rate their feelings of hunger in advance of the scanning procedure and afterwards, using a Visual Analogue Scale (VAS) which consisted of a 10cm line, with ‘not hungry’ and ‘extremely hungry’ as anchors. Subjects were asked to indicate their score on the line, higher scores indicating a more hungry feeling. Additionally, 30 minutes after ingestion after finishing the MRI procedure, pleasantness and sweetness of the drink were rated using a similar VAS with ‘disgusting’ to ‘very tasty and ‘not ‘sweet’ to ‘extremely sweet’ as anchors.

MRI data acquisition

Magnetic Resonance Imaging was performed using a 3 Tesla Achieva whole body MRI scanner (Philips Healthcare, Best, The Netherlands) equipped with a 32-channel head

coil. The protocol for structural MRI comprised a scout view for planning, a high resolution 3D T₁-weighted sequence for registration purposes and a mid-sagittal high resolution single slice for accurate hypothalamus and VTA localization (repetition time 550ms, echo time 10 ms, field of view 208 x 208 mm, voxel size = 0.52 x 0.52 x 14 mm). Mid-sagittal fMRI was performed for 21 minutes in total, by a T₂* weighted, gradient echo-planar imaging (EPI) sequence mid-sagittal single slice that renders BOLD contrast (repetition time 120 ms; echo time 30ms; flip angle 30°; field of view 208 x 208 mm; voxel size = 0.81 x 0.90 x 14 mm; scanning time of one dynamic image 2.52 seconds for a total of 500 data points). A slice thickness of 14 mm was chosen to encompass the hypothalamus in the left to right direction and a single slice technique was used for sufficient signal to noise ratio.

fMRI processing

Imaging data were pre-processed and analyzed using Functional Magnetic Resonance Imaging of the Brain Software Library (FSL) version 5.0.8. [30]. The mid-sagittal fMRI was preprocessed as described before [31]. In short, all 500 dynamic images were motion-corrected by registration to the image that was acquired halfway through the fMRI period (scan 250) by means of Multimodality Image Registration using Information Theory (MIRIT) software in FSL by maximization of mutual information [32]. The complex data were averaged for each set of 4 subsequent volumes after which 125 images were rendered. Regions of interests (ROIs) were drawn manually, using subject- specific T₁-weighted images as a visual aid to define anatomical borders. Three ROIs were drawn: the hypothalamus, the VTA and a reference area. The ROI for the hypothalamus was drawn as described earlier using the optic chiasm, mammillary bodies, thalamus and anterior commissure as anatomical landmarks [31]. Using literature describing the VTA region [33, 34] we defined the ROI for the VTA in the midbrain with half the volume of the hypothalamus. The top of the cerebral aqueduct and the mammillary bodies were used as anatomical landmarks (figure 1). To correct for scanner drift, a third internal reference ROI half the volume of the hypothalamus ROI was selected in the gyrus superior to the genu of the corpus callosum in the grey matter. Hypothalamic and VTA BOLD signals were adjusted linearly to the BOLD signal of this internal reference ROI. Subsequently, to investigate the individual BOLD response after study intervention on the hypothalamus and VTA, the BOLD response after ingestion were all normalized to the BOLD response before ingestion, i.e. the mean BOLD signal before ingestion; the average BOLD signal of the first 4 minutes of the fMRI scan (figure 2).

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Statistical analysis

Statistical testing of hypothalamus and VTA activations was performed using SPSS version 23. Differences in VAS scores and blood values between pre and post-ingestion and between conditions were tested using repeated measures ANOVA. To test for differences in the post-ingestion BOLD responses between sugars and sweeteners and the water control a mixed model approach was used, with Intervention and Study visit number as fixed effects and Time point as a covariate and a random effect for Subject*Study visit number. To determine the time effects in the BOLD response the entire 12 minute post-ingestion response was divided into four arbitrary 3-minute time blocks. Mixed model analysis for comparison between sugars and sweeteners and the water control was performed per time block. Significances are presented uncorrected. Pearson correlations were used to determine the association between BOLD response and blood levels and VAS scores.

Figure 1. A representative sagittal view with the hypothalamus (A) and the Ventral Tegmental Area (VTA) (B) ROIs. For the hypothalamus ROI (A), the optic chiasm (1), the mammillary bodies (2), the thalamus (3)

and the anterior commissure (4) were used as landmarks. For the VTA ROI (B), the top of the cerebral aqueduct (5) and the mammillary bodies (2) were used as landmarks. The reference ROI was drawn superior of the genu to the corpus callosum (6) in the grey matter.

RESULTS

Blood glucose and insulin levels

Blood glucose and insulin level measurements for all participants are shown in table 2. All subjects demonstrated normal fasting glucose levels and the average fasting glucose was not different between conditions. As expected, glucose and sucrose ingestion led to significant increases in both blood glucose and insulin levels. Fructose had no significant effect on blood glucose levels but led to a small significant increase in blood insulin levels. Sucralose had no significant effect on blood glucose levels but led to a small yet significant decrease in insulin levels.

Visual analogue scores for hunger and subjective rating of the study stimuli

VAS scores for hunger and subjective rating of the stimuli for all participants per study stimulus are shown in table 3. No significant differences were found between the stimuli in hunger score before ingestion. After ingestion of water the VAS score and delta VAS score for hunger was significantly increased compared to the other or stimuli. The taste of sucralose and water was rated significantly less pleasant compared to fructose and sucrose and as expected the water condition was rated significantly less sweet compared to the sugars and sweetener. All other study stimuli were rated similarly for sweetness.

Hypothalamic BOLD response

Figure 2 shows the mean normalized hypothalamic BOLD response curves after ingestion of the five study stimuli. Table 4 shows the corresponding BOLD effect sizes and statistical differences relative to the control condition, both for the entire response

Table 2. blood glucose and insulin levels

Glucose Fructose Sucrose Sucralose Water

Pre-ingestion glucose 5.0 ± 0.7 4.9 ± 0.4 4.8 ± 0.3 5.2 ± 1.6 4.7 ± 0.9 Post-ingestion glucose 7.0 ± 1.1* 5.4 ± 0.7 7.0 ± 0.7* 4.7 ± 0.8 4.6 ± 0.9 Delta glucose 2.0 ± 1.4 0.5 ± 0.9 2.3 ± 0.8 -0.5 ± 1.7 -0.1 ± 0.5 Pre-ingestion insulin 6.4 ± 4.7 6.5 ± 4.0 7.5 ± 6.0 6.5 ± 5.3 8.0 ± 5.4 Post-ingestion insulin 32.5 ± 20.3* 12.3 ± 5.9* 26.5 ± 18.9* 5.3 ± 4.6* 6.6 ± 5.1 Delta insulin 26.0 ± 18.5 5.8 ± 3.9 19.0 ± 15 -1.6 ± 1.6 -1.5 ± 3.6 All values in mean± standard deviation. Glucose levels in mmol/L, insulin levels in mU/L. *significantly different between pre and post-ingestion at p<0.05.

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curve and the 3-minute response intervals. Ingestion of plain water led to an increase in BOLD signal, whereas all sugars and sucralose led to a decrease in BOLD signal. Compared to the control condition, glucose led to an immediate and continues decrease in BOLD response (mean difference 1.8%, p=0.0003). Fructose and sucrose also led to a significant average decrease compared to the water control condition (mean difference 1.2%, p=0.006 and mean difference 1.3%, p=0.003 respectively). However, the response in the first 3 minutes after ingestion of both fructose and sucrose was not significantly different from water, indicating a delayed response to these stimuli. The response to sucralose compared was significantly lower (mean difference 0.9%, p=0.026), and did not increase over time as was the case with the other sugars.

Ventral Tegmental Area BOLD response

Figure 3 shows the mean normalized BOLD response for all participants in the VTA over time after ingestion of the study stimuli. In the VTA, all conditions led to an increase in BOLD response after ingestion. Table 5 shows the mean effect sizes and statistical differences relative to the control condition over the entire 12 minute post ingestion period and during the four 3-minute time blocks. Ingestion of plain water

Table 3. Visual Analogue Scale scores for hunger, pleasantness and sweetness

Glucose Fructose Sucrose Sucralose Water

Pre-ingestion VAS hunger 5.3 ± 2.5 5.7 ± 1.9 4.8 ± 2.1 5.3 ± 1.9 5.2 ± 1.9 Post-ingestion VAS hunger 5.1 ± 2.4 5.5 ± 1.9 5.0 ± 1.8 5.3 ± 2.3 5.8 ± 2.3* Delta VAS hunger -0.2 ± 1.5 -0.2 ± 2.3 0.3 ± 1.8 0.0 ± 1.2 0.6 ±0.9* VAS pleasantness 5.9 ± 1.7 6.8 ± 1.8 6.4 ± 1.9 5.3 ± 2.1* 5.1 ± 1.1* VAS sweetness 5.4 ± 2.1 6.4 ± 1.4 6.9 ± 1.4 5.6 ± 2.2 0.8 ± 1.6* All values in mean± standard deviation *significantly different form other study conditions at p<0.05.

Table 4. Mean difference in post-ingestion hypothalamic BOLD responses to the study stimuli and the water

control condition

Average

response Response min 9-11 Response min 12-14 Response min 15-17 Response min 18-21

Glucose -1.9 ± 0.4* -1.4 ± 0.5* -1.5 ± 0.4* -1.9 ± 0.5* -2.0 ± 0.5* Fructose -1.2 ± 0.4* -0.5 ± 0.5 -1.2 ± 0.4* -1.6 ± 0.5* -1.5 ± 0.5* Sucrose -1.3 ± 0.4* -0.9 ± 0.5 -1.3 ± 0.5* -1.3 ± 0.5* -1.6 ± 0.5* Sucralose -0.9 ± 0.4* -0.9 ± 0.5* -1.2 ± 0.4* -1.2 ± 0.4* -0.9 ± 0.5* Mean difference in BOLD response indicated in mean % change ± SE. *significantly different form the water control condition at p<0.05.

led to an increase in BOLD signal, comparatively all sugars led to a smaller increase in BOLD signal (mean difference 0.4%). However, when comparing the response to the sugars to the response to plain water, no significant differences were found. The response to sucralose showed a close resemblance to the response to water, as indicated by the smallest mean difference between the water and sucralose response (mean difference 0.1%). The response to sucralose, and also to water, remained elevated over the entire 12 minute post-ingestion time period whereas the response to the natural sugars appeared transient. Additionally, glucose ingestion was the only stimulus that led to an initial decrease in VTA BOLD response in the first 3 minutes after ingestion.

Figure 2. Normalized BOLD response (% change) in the hypothalamus after ingestion of water (control condition), glucose, fructose, sucrose and sucralose stimuli. Data presented as mean response for all

participants (n=16) per minute with standard error. Post ingestion response was calibrated to the baseline period. Grey bar represents ingestion period, data recorded during this period was excluded from analysis.

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Correlation of hypothalamic and VTA BOLD response with VAS and blood levels

We found a significant negative correlation between the rating of perceived sweetness and the hypothalamic BOLD response (Pearson correlation coefficient= -0.350, p=0.002). This indicates that a sweeter rated stimulus leads to a stronger decrease in hypothalamic response. We found no significant correlations between blood glucose and insulin levels and the BOLD responses of neither the hypothalamus nor the VTA.

DISCUSSION

Our data show that the hypothalamus and VTA demonstrate different BOLD responses to glucose, fructose, sucrose and sucralose ingestion. In contrast to glucose, which has a direct and effect on the hypothalamus, fructose and sucrose ingestion resulted in delayed and smaller hypothalamic BOLD responses. Sucralose ingestion led to a transient response in the hypothalamus. In the VTA, sucralose ingestion led to the same effect as the ingestion of plain water, being a prolonged activation over the measured time period. On the contrary, the natural sugars glucose, fructose and